Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are also activated by target MHC I. The mouse Ly-49D receptor is structurally not an inhibitory receptor and thus our studies have been focused on the ability of select Ly-49 receptors to activate NK cytotoxicity and function. Since Ly-49D does not become tyrosine phosphorylated upon activation, we have demonstrated that specific Ab cross-linking, coprecipitates a 16-kDa tyrosine- phosphorylated protein (pp16), that was cloned in the human and is termed DAP12. Association of DAP12 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49D[R54L]) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49D(R54L) transfectants fail to mediate Ca2+ mobilization following Ab cross- linking. Therefore, signaling through Ly-49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families. To define Ly-49D-ligand interactions, we utilized both transfected rat NK cell lines and primary mouse subests. RNK-16 expressing Ly-49D (RNK.mLy-49D) were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy- 49D cells lysed YB2/0. Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb and this augmented lysis was specifically inhibited by F(ab)2 anti-Ly-49D and F(ab)2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. Primary cells were analyzed using sorted NK cell subsets as effectors and a panel of 51Cr- labeled Con A lymphoblasts as targets in the presence or the absence of Abs to Ly49 and/or class I molecules. Our results demonstrate that the activating receptor Ly49D delivers stimulatory signals for target cell lysis upon interacting with H2-Dd, Dr, and Dsp2, but not H2b or H2k class I Ags. Thus, lysis of class I MHC-bearing targets by NK cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly-49 receptors. Activation of NK cells by nonself class I molecules was not predicted by the missing self hypothesis. NK cells mediate the specific rejection of bone marrow cell (BMC) allografts in lethally irradiated mice. Depletion of Ly-49D+ NK cells in H-2b mice abrogated their ability to reject H-2d BMC allografts. Similarly, Ly- 49C+ NK cells also were shown to mediate the specific rejection of H-2d BMC. When both subsets were depleted, an additive enhancement of BMC engraftment was observed, indicating that both subsets play a role in the rejection of allogeneic H-2-homozygous H-2d BMC. However, rejection of H-2(b x d) or D8 (H-2b, Dd transgene) BMC allografts was unaffected by Ly-49C+ NK cell depletion in H-2b mice. In marked contrast, depletion of Ly-49D+ NK cells in H-2b mice totally abrogated the rejection of H-2(b x d) heterozygous BMC in support of in vitro data suggesting that Ly-49D+ NK cells receive activating signals. Therefore, NK subsets demonstrate a differential ability to reject H-2 homozygous and heterozygous BMC. - Class I, Ly-, , mouse, NK, receptor, regulation,